Chemical reagents and cell culture
Cell culture medium and antibiotics were purchased from WELGENE Inc. (Daegu, Korea). Fetal bovine serum (FBS) was purchased from Lonza (Basel, Switzerland), and other experimental reagents were obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA), unless otherwise specified.
RP cells were isolated from the palatal tissues of 5-week old male Sprague–Dawley (SD) rats. Isolated palatal tissues were washed with phosphate buffered saline (PBS) (PH 7.4), aseptically minced into pieces, and then immersed in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and antibiotic solution (100 U/ml of penicillin-G and 100 μg/ml of streptomycin) at 37 °C in a humidified incubator (5% CO2/95% air). After 20 days of culture with medium changes at 3-day intervals, RP cells were collected and sub-cultured under the same conditions. Passages three through five were used for this study. Although we did not identify the type of palatal cells at the molecular level, morphological features of the isolated cells under the microscope indicated distinct dominance of fibroblast-like cells.
RP cells incubated until 80% confluent were removed and sub-cultured at 0.8 × 105 cells per ml in 96-well plates and incubated at 37 °C with 5% CO2 in air for 24 h, and then the cells were treated with DMOG at various concentrations ranging from 0.1 to 10 mM. After treatment for 24 h, the cell viabilities were quantified using the Cell Counting Kit-8 (WST-8) (Dojindo Laboratories, Kumamoto, Japan). Cells were incubated in 100 μl of WST-8 solution for 1 h at 37 °C in a humidified atmosphere (5% CO2/95% air). The absorbance was measured at a wavelength of 450 nm using a plate reader (Sunrise, TECAN, Salzburg, Austria). The optical density of untreated cells was designated as 100%, and cell viability of treated cells was expressed as the percentage of untreated negative control.
Cell migration assay
For the in vitro cell migration assay, a culture insert (ibidi GmbH, Martinsried, Germany) was used to create a wound in cell culture. The culture insert was placed on a culture dish, and 70 μl of RP cell suspension (5 × 105 cells/ml) was added into both wells of the insert. The RP cells were incubated at 37 °C for 48 h and then exposed to DMOG (0, 0.1, 0.5, 1, 2 mM) in culture media containing 2% FBS for the cell migration analysis. Wound closure was observed and recorded at intervals under a phase contrast microscope (Olympus, Tokyo, Japan). To quantify cell migration, the uncovered area where no cells were present was measured by using ImageJ program, and the ratio of uncovered area between untreated control and treated groups was obtained.
mRNA expression analysis by real-time PCR
The effect of DMOG on the expression of VEGF mRNA was investigated by real-time polymerase chain reaction (RT-PCR) assay. After treatment with DMOG at 0, 0.1, 0.5, 1, and 2 mM for 24 h, total RNA was isolated using RNA extraction reagent (WelPrep Total RNA Isolation Reagent, WELGENE Inc.). From the total RNA, cDNA was prepared using a cDNA synthesis kit (Power cDNA Synthesis Kit, iNtRON Biotechnology, Sungnam, Korea), and RT-PCR was performed in an ABI PRISM 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with 20 μl reaction volumes containing 10 μl SYBR premix Ex Taq (Takara Bio, Otsu, Japan), 0.4 μl ROX Reference Dye II (Takara Bio), cDNA, and primers. The primers for gene amplification were as follows: VEGF sense, 5’-GAGTATATCTTCAAGCCGTCCTGT-3’; VEGF antisense, 5’-ATCTGCATAGTGACGTTGCTCTC-3’; GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) sense, 5’-TGTGTCCGTCGTGGATCTGA-3’; GAPDH antisense, 5’-CCTGCTTCACCACCTTCTTGAT-3’. The PCR conditions were 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 63 °C (34 s) for VEGF. All reactions were run in triplicate. Gene expression was evaluated on the basis of the threshold cycle (CT value) and normalized to the expression of the GAPDH gene.
Western blot analysis
Western blot analysis was performed to examine the protein expression of HIF-1α and VEGF in DMOG-treated palatal cells. After treatment with DMOG at various concentrations for 24 h, cells were lysed in extraction buffer containing 50 mM Tris base-HCl (PH 7.5), 150 mM NaCl, 0.5% Triton-X 100, and one tablet of protease inhibitor cocktail (1 tablet/10 ml, Roche Applied Science, Mannheim, Germany) for 45 min on ice. Extracts containing equal amounts of protein were run on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were incubated with rabbit polyclonal antibodies against VEGF, HIF-1α, or GAPDH in PBST (PBS containing 0.1% Tween 20) for 1.5 h, washed three times with PBST, and then probed with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase. The protein bands were visualized using a chemiluminescence kit (WEST-ZOL plus Western Blot Detection System, iNtRON Biotechnology). Chemiluminescence was detected using the LAS 1000 Plus Luminescent Image Analyzer (Fuji Photo Film, Tokyo, Japan). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Rat palatal wound healing assay
After confirming the angiogenesis effect of DMOG on RP cells, the effect of DMOG on wound healing of palatal tissue was performed in a rat palatal wound model. All rats were housed under specific pathogen-free (SPF) conditions at the animal experimental center of Seoul National University Dental School. Eighteen 13-week-old male SD rats (six rats for each group), weighing 300–350 g, were used in the present study. All animal experiments were approved by the Institutional Animal Care and Use Committee (SNU-130123-8-1) of Seoul National University (Seoul, Korea). Under general anesthesia, punch wounds were made on a central area of hard palate with a disposable 3-mm diameter biopsy punch (Kai Industries Ltd., Gifu, Japan), exposing a circular area of bare bone. Hyaluronic acid (HA) ointment (20 mg/ml) containing 0, 0.5, or 1 mg/ml (5.7 mM) DMOG was applied to the wound area. After the surgery, animals were fed a standard diet of pellets and water with enrofloxacin. The agents were re-applied on days 2 and 4 under anesthesia to reduce stress, and the rats were sacrificed on day 7. The hard palate was separated, and the wound area was observed with a stereoscopic microscope (Nikon, Tokyo, Japan) and by histological analysis. The wound area on the microscopic images was calculated using CellSense Dimension 1.6 software (Olympus, Tokyo, Japan). Palatal specimens were taken for histomorphometric evaluation and samples were stained with haematoxylin and eosin (H&E) staining. More than ten slides for each sample were prepared, and we selected a section that exhibited the widest diameter of wound for histological analysis. The sections were examined under a light microscope (Olympus), and the distance of wound margins in each section was measured with a calibrated ocular micrometer.
For statistical analysis, each experiment was performed in triplicate unless otherwise specified. The data are expressed as the mean ± Standard Deviation. Statistical analyses were determined by one-way ANOVA for in vivo study and cell migration assay. The unpaired Student’s t-test was used for the other in vitro results. A P-value < 0.05 was considered statistically significant.